Increased expression of chitinase 3-like protein 1 secondary to joint immobility.

Previous studies have established mechanical stimulation of joints is necessary to maintain the structure and function of the articular cartilage. Immobilization of the rat knee joint induces cartilage degeneration and reduces the joint range of motion, two of the clinical parameters used to define a joint contracture. We hypothesized chondrocytes from articular cartilage increase their expression of the chitinase 3-like protein 1 (CHI3L1) gene in response to joint immobility. We selected the CHI3L1 gene on the basis of its identification as a differentially expressed gene in the articular cartilage obtained from immobilized rat knee joints. Expression of CHI3L1 mRNA was increased after 2 and 4 weeks of immobility. A time-course study revealed that CHI3L1 immuno-reactivity was increased at 2 and 4 weeks and return to basal levels at all later time points. CHI3L1 gene adds to the list of differentially expressed genes defining the response of cartilage to joint immobility. Our data confirm a protective role for CHI3L1 in the initial phase of degeneration induced by immobility.

HC-gp39 contributes to chondrocyte differentiation by inducing SOX9 and type II collagen expressions.

OBJECTIVE: The transcription factor SOX9 has been shown to be linked to chondrocyte differentiation and induction of type II collagen synthesis. Since the chitinase-like protein, human cartilage glycoprotein 39 (HC-gp39), can be expressed by articular chondrocytes and has been shown to enhance chondrocyte mitogenesis through MAP kinase and PI3 kinase-mediated signalling, we hypothesized that it may also promote synthesis of cartilage matrix components through induction of SOX9, utilizing similar signalling pathways. METHODS: Primary chondrocytes from neonatal mouse rib cartilage were exposed to purified HC-gp39. The response of the cells was evaluated in terms of SOX9 induction and synthesis of type II collagen. Signalling pathways activated following HC-gp9 exposure were analyzed by Western blotting of cell lysates with phosphorylation-specific antibodies as well as by using selective inhibitors. RESULTS: HC-gp39 induced both SOX9 and type II collagen synthesis. Similar results were observed for IGF-1. This process required signalling through both MAP kinase and PI3 kinase pathways resulting in rapid phosphorylation of ERK1/2 and AKT, respectively. Neither HC-gp39 nor IGF-1 induced activation of SAPK/JNK. CONCLUSIONS: The effects of HC-gp39 on chondrocyte function suggest that this molecule may promote the maintenance or expression of a chondrocytic phenotype. Its expression in injured or degenerate cartilage could be related to the initial repair-response and increased matrix synthesis observed in osteoarthritic cartilage.

Differential regulation and expression of hyaluronan synthases in human articular chondrocytes, synovial cells and osteosarcoma cells.

Recently three isoforms of hyaluronan synthase (HAS), the enzyme responsible for hyaluronate/hyaluronan (HA) biosynthesis, have been cloned, allowing us to study their expression pattern. Our objective was to determine which of the HAS isoenzymes were expressed in human articular chondrocytes, synovial fibroblasts and osteosarcoma cells, whether their expression could be modulated by growth factors (insulin-like growth factor-1, basic fibroblast growth factor and transforming growth factor (TGF-beta1) and cytokines [interleukin 1beta1 (IL-1beta)], and whether changes in the rate of HA synthesis by the cells correlated with changes in mRNA levels for one or more of the HAS isoforms. All three HAS isoforms were found to be expressed in the cultured cells analysed in this study, although the relative proportions varied for each cell type. HAS2 mRNA was usually predominant in chondrocytes, whereas synovial cells contained increased amounts of HAS1. HAS3 was always the least abundant message. The rapidly growing osteosarcoma cells contained almost exclusively HAS2 message. HAS usage in uncultured cartilage and synovial tissues was similar to that in the cultured cells, with HAS2 message being the predominant species in cartilage and HAS1 usually being the predominant species in synovium. HA synthesis was stimulated by the growth factors, but the extent of the response was cell-type specific. Synovial cells responded particularly well to IL-1beta, and showed a unique synergistic response when IL-1beta was used in combination with TGF-beta1. This response was much reduced in articular chondrocytes and absent in the osteosarcoma cells. Analysis of changes in HAS message levels indicated that there was often no correlation with the changes in HA secretion following exposure to growth factors. Although HAS-1 mRNA was increased in synovial cells after exposure to TGF-beta1/IL-1beta, the magnitude of the change was far less than the effect on HA synthesis. Our data thus suggest that HAS gene usage is tissue specific, and the regulation by growth factors is unique for each HAS gene and is further modulated by cell-specific factors. In addition, regulation of HA biosynthesis appears to be multi-faceted, with control of HAS gene expression and mRNA levels being only one aspect of this process.

Bone morphogenetic protein-1 processes probiglycan.

Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays important roles in regulating the deposition of fibrous extracellular matrix in vertebrates, including provision of the procollagen C-proteinase activity that processes the major fibrillar collagens I-III. Biglycan, a small leucine-rich proteoglycan, is a nonfibrillar extracellular matrix component with functions that include the positive regulation of bone formation. Biglycan is synthesized as a precursor with an NH(2)-terminal propeptide that is cleaved to yield the mature form found in vertebrate tissues. Here, we show that BMP-1 cleaves probiglycan at a single site, removing the propeptide and producing a biglycan molecule with an NH(2) terminus identical to that of the mature form found in tissues. BMP-1-related proteases mammalian Tolloid and mammalian Tolloid-like 1 (mTLL-1) are shown to have low but detectable levels of probiglycan-cleaving activity. Comparison shows that wild type mouse embryo fibroblasts (MEFs) produce only fully processed biglycan, whereas MEFs derived from embryos homozygous null for the Bmp1 gene, which encodes both BMP-1 and mammalian Tolloid, produce predominantly unprocessed probiglycan, and MEFs homozygous null for both the Bmp1 gene and the mTLL-1 gene Tll1 produce only unprocessed probiglycan. Thus, all detectable probiglycan-processing activity in MEFs is accounted for by the products of these two genes.

Regulation of cartilage oligomeric matrix protein synthesis in human synovial cells and articular chondrocytes.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a component of the extracellular matrix of articular cartilage. Its increased presence in synovial fluid and serum has been associated with accelerated joint damage in patients with rheumatoid arthritis (RA) and osteoarthritis. To fully understand the reasons for fluctuations of COMP levels, we studied the biosynthesis of this molecule in cells derived from joint tissues. METHODS: Synovial cells were derived from synovial tissues of patients with RA, and human articular chondrocytes were prepared from normal articular cartilage. Analysis by Northern blotting was used to evaluate steady-state levels of COMP messenger RNA (mRNA), while secretion of the protein into culture media was analyzed by Western blotting. Expression of COMP in synovial tissues was studied by reverse transcriptase-polymerase chain reaction analysis and by in situ hybridization. RESULTS: COMP was synthesized and secreted by synovial cells as well as by articular chondrocytes in culture. The basal rate of synthesis was very low; however, COMP biosynthesis in both cell populations was induced very strongly by transforming growth factor beta1 (TGFbeta1). Interleukin-1beta counteracted COMP induction by TGF-beta1. COMP was not detected in culture media of skin or fetal lung fibroblasts, either in the absence or the presence of TGFbeta1. COMP mRNA was also present in fresh synovial tissue specimens obtained from patients with RA. CONCLUSION: COMP is synthesized and secreted not only by articular chondrocytes, but also by synovial fibroblasts. The demonstration of COMP expression in surgical specimens of synovial tissues suggests that the inflamed synovium may provide an additional source for the elevated levels of COMP observed in arthritis. Thus, increased COMP levels in body fluids may be indicative of active synovitis as well as of accelerated joint erosion.

The gene organization, chromosome location, and expression of a 55-kDa matrix protein (PRELP) of human articular cartilage.

The gene corresponding to a 55-kDa matrix protein previously described in adult human articular cartilage was characterized by sequencing of genomic clones. The deduced protein sequence corresponds to the recently described matrix protein PRELP. The protein was encoded by messages of 1.7, 4.6, and 6.7 kb, whose relative abundance increased as their size decreased. The message heterogeneity appears to originate from variation in the length of the 3'-untranslated region, with the smallest message being contained within the reported sequence and the larger messages having extended 3'-untranslated regions. Two introns were identified within the genomic sequence encoding the smallest message. The first intron of about 6.7 kb resides 16 nucleotides prior to the translation initiation codon, and the second intron of about 2.6 kb resides 173 nucleotides prior to the translation termination codon. The gene, which encompasses at least 16 kb of genomic DNA, was shown to reside on chromosome 1q32. Primer extension techniques were used to establish that the coding sequence commences 199 bp downstream from the major transcription start site. Analysis of the DNA sequence upstream from the transcription start site reveals the presence of numerous potential transcription factor binding sites, but no CAAT or TATA box. At the message level, gene expression was at a high level in juvenile and adult cartilage, but not in the fetus or neonate. The presence of protein in the cartilage matrix was also much lower in the neonate than in the adult. In noncartilagenous tissues appreciable message levels were observed only in the adult lung.

Modulation of the catabolic effects of interleukin-1 beta on human articular chondrocytes by transforming growth factor-beta.

The effects of IL-1 beta and TGF-beta on the biosynthesis of extracellular matrix structural components relative to the metalloproteinases and their inhibitor TIMP1 in human articular chondrocytes were investigated. It has been proposed that TGF-beta, acting as a positive regulator of matrix accretion, can counteract the increased loss of cartilage matrix induced by IL-1 beta. To allow a comparison of their effects on mRNA levels for these different components, quantitation by competitive RT/PCR was employed. This method was found to give reproducible estimates of mRNA levels and the observed effects of IL-1 beta and TGF-beta on individual components of this system agree with qualitative data obtained by northern blotting. IL-1 beta had a more pronounced effect on aggrecan mRNA levels than on those for type II collagen. Similar quantitative differences were observed between collagenase and stromelysin mRNA levels. TGF-beta generally counteracted the effects of IL-1 beta, and new steady state levels were attained within 24 h. However, the reversal of IL-1 beta induced suppression of matrix protein mRNA levels appeared more effective than its suppression of the increase in stromelysin and collagenase mRNA levels. Similarly TGF-beta did not reduce the extent of IL-1 beta induced secretion of stromelysin at the protein level. TIMP1 mRNA levels were only slightly reduced by IL-1 beta; however this cytokine effectively suppressed its induction by TGF-beta. The higher concentrations of TGF-beta and longer exposure times required to overcome the suppressive effects of IL-1 beta suggest that the interaction between IL-1 beta and TGF-beta in the regulation of TIMP1 expression follows a different mechanism to that operating for the metalloproteinases and matrix proteins. Thus the overall potential of TGF-beta to inhibit proteolysis is attenuated by its much slower effect on TIMP1 mRNA levels.

Chondrogenesis in periosteal explants. An organ culture model for in vitro study.

Periosteal grafts have chondrogenic potential and have been used to repair defects in articular cartilage. We studied the effects of the culture conditions and of transforming growth factor-beta 1 on chondrogenesis in rabbit periosteal explants that were cultured in vitro. A total of 390 periosteal explants were obtained from the anteromedial sides of the proximal parts of the tibiae of eleven rabbits that were two weeks, two months, or six months old. The culture medium (alpha minimum essential medium or Dulbecco minimum essential medium) contained fetal calf serum, with or without transforming growth factor-beta 1, at a concentration of one or ten nanograms per milliliter for the first two weeks of culture. Three hundred and twenty-one explants were submerged in liquid medium and sixty-nine were suspended in an agarose gel; they were then evaluated histochemically, histomorphometrically, and by collagen-typing. In the media without agarose, in the presence of ten nanograms of transforming growth factor-beta 1 per milliliter, chondrogenesis was commonly seen after two to four weeks with use of safranin-O staining and histomorphometry. In the agarose gels, chondrogenesis from the periosteum was observed at four and six weeks and was enhanced by the presence of one or ten nanograms of transforming growth factor-beta 1 per milliliter. The combination of agarose with transforming growth factor-beta 1 most favored the formation of cartilage, which was maximum at six weeks in the presence of ten nanograms of transforming growth factor-beta 1 per milliliter. Under these conditions, chondrogenesis occurred in almost every explant, with 50 +/- 30 per cent of the tissue being composed of cartilage. Type-II collagen was present in the explants that had undergone chondrogenesis.

Changes in the expression of decorin and biglycan in human articular cartilage with age and regulation by TGF-beta.

In chondrocytes isolated directly from human articular cartilage, without subsequent culture, biglycan mRNA levels decreased with the age of the donor, whereas those for decorin increased. In cultured chondrocytes in the presence of FCS, mRNA levels for biglycan remained similar to those in uncultured counterparts, while decorin transcription became depressed. The differential effect on decorin and biglycan mRNA expression was mimicked by the addition of TGF-beta. In the absence of exogenously added growth factors (TGF-beta or FCS) biglycan mRNA levels decreased, while those for decorin increased. In contrast, IGF-I showed no differential modulation of the relative abundance of the two messages. The opposite regulation of these two proteoglycans by TGF-beta was also reflected at the level of protein synthesis. It would appear that the need of articular cartilage for decorin is greatest in the adult, whereas the need for biglycan is greatest in the juvenile.

Human cartilage gp-39, a major secretory product of articular chondrocytes and synovial cells, is a mammalian member of a chitinase protein family.

One of the major secreted proteins of human articular chondrocytes in monolayer or explant culture and of synovial fibroblasts is a glycoprotein with an apparent molecular weight of approximately 39,000, referred to as human cartilage glycoprotein-39 (HC gp-39). The protein was purified, and its complete cDNA sequence was determined. It contained an open reading frame coding for a 383-amino acid long peptide. Comparison of the deduced amino acid sequence with known sequences revealed that HC gp-39 contained regions displaying significant homology with a group of bacterial and fungal chitinases and a similar enzyme found in the nematode, Brugia malayi. In addition significant homologies were observed with three mammalian secretory proteins of as yet unknown function, suggesting that a related protein family exists in mammals. The human protein does not possess any glycosidic activity against chitinase substrates, arguing against any function as an endoglycosidase with specificity for N-acetylglucosamine. Analysis by Northern blotting and by reverse transcription/polymerase chain reaction showed mRNA for HC gp-39 to be present in human articular chondrocytes as well is in liver, while mRNA was undetectable in muscle tissues, lung, pancreas, mononuclear cells, or fibroblasts. Neither the protein nor mRNA for HC gp-39 was detectable in normal newborn or adult human articular cartilage obtained at surgery, while mRNA for HC gp-39 was detectable both in synovial specimens and in cartilage obtained from patients with rheumatoid arthritis. These observations suggest that the expression of HC gp-39 may be related to a response of these cells to an altered tissue environment.

Induction of synthesis and release of interleukin-8 from human articular chondrocytes and cartilage explants.

OBJECTIVE: The activation of neutrophils in the joint space may contribute to the destruction of cartilage matrix observed in rheumatoid arthritis. The capacity of articular chondrocytes to synthesize and secrete interleukin-8 (IL-8) and GRO alpha, two potent neutrophil chemoattractant peptides, was investigated to determine whether cartilage itself could serve as a source of these small cytokines. METHODS: Induction of IL-8 and GRO protein was studied both at the messenger RNA (mRNA) and the protein level by reverse transcriptase/polymerase chain reaction and metabolic labeling, respectively. RESULTS: Strong induction of IL-8 was observed in primary cultures of articular chondrocytes as well as in cartilage explants stimulated with IL-1 beta. The increased secretion of the IL-8 protein was accompanied by corresponding increases in mRNA levels. In contrast to other connective tissue cells, a peptide corresponding in molecular size to the GRO proteins was only weakly induced in cartilage explants or primary chondrocyte cultures. However, mRNA for all 3 members of the GRO family was easily detectable in cultured chondrocytes following stimulation with IL-1 beta. In explanted cartilage, mRNA for only GRO gamma was found to be induced. Newly synthesized IL-8 was slowly released from cartilage explants over a prolonged time in culture. CONCLUSION: The results suggest that synthesis and secretion of the diverse members of the IL-8/GRO family is regulated in a tissue-specific or cell-specific manner. The slow release of IL-8 from articular cartilage following induction by IL-1 beta could establish a chemotactic gradient toward the articular surface and mediate the migration and attachment of neutrophils and lymphocytes to this tissue.

Phospholipase A2 is a major component of the salt-extractable pool of matrix proteins in adult human articular cartilage.

Adult human articular cartilage contains a component with an apparent molecular weight of 16 kd, which is extractable with high ionic strength buffers. This protein, which, in addition to lysozyme, is one of the most prominent components in salt extracts of adult cartilage, is not detectable in cartilage from newborns. We performed N-terminal sequence analysis to identify the protein. The amino acid sequence obtained for the first 20 residues was identical to that reported for phospholipase A2 (PLA2) from human placenta and human synovial cells. The extractable PLA2 was found to be active. The lack of PLA2 in salt extracts from newborn cartilage observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis was confirmed by the very low levels of PLA2 activity detectable in these preparations. PLA2 was clearly present in cartilage extracts from an 18-year-old subject and a 19-year-old subject, suggesting that its accumulation begins at some stage during the adolescent growth period. The enzyme does not appear to be released from cartilage matrix under normal physiologic conditions, and it is possible that the accumulation of PLA2 in maturing cartilage is a result of the decreased matrix turnover associated with the termination of skeletal growth. Whether PLA2 is active in the cartilage matrix, its precise localization, and its effects on the resident chondrocytes remain to be determined.

Interrelationship of active and latent secreted human cathepsin B precursors.

Two high-Mr forms of cathepsin B have been described previously, both of which are stable at alkaline pH, in contrast with the lysosomal proteinase. One form is latent and activated by pepsin treatment; the other form is active as measured with synthetic substrates. In the present study it was shown that the two forms are indistinguishable on the basis of molecular size as determined by gel-filtration chromatography or sodium dodecyl sulphate/polyacrylamide-gel electrophoresis followed by immunoblotting. Both forms lose their alkali-stability upon exposure to Hg2+, and after Hg2+ treatment the latent form becomes immuneprecipitable by an antiserum that reacts only with denatured cathepsin B. Lysosomal cathepsin B is bound by the plasma proteinase inhibitor alpha 2-macroglobulin, a process that requires proteolytic cleavage of the inhibitor. In contrast, the stable active form of cathepsin B is not bound by this inhibitor unless this enzyme is first destabilized by Hg2+ treatment. These results indicate that cathepsin B exists in three different states of activity, completely latent, partially active and fully proteolytically active. To exhibit true endopeptidase activity it seems that the enzyme must be in an alkali-unstable form.

Rat mammary gland in culture secretes a stable high molecular weight form of cathepsin L.

Culture medium from rat mammary gland explants was analyzed for the presence of cysteine proteinases. In addition to a putative precursor of the lysosomal enzyme cathepsin B, a cysteine proteinase with enzymatic properties similar to those reported for cathepsin L was found. Further evidence of the cathepsin L-like nature of this activity was provided by its high sensitivity towards the diazomethane inhibitors Z-Phe-Phe-CHN2 and Z-Phe-Ala-CHN2 and towards leupeptin. The secreted form of cathepsin L is distinguished from the lysosomal form by its increased stability at alkaline pH and by its larger molecular size. It may thus represent an incompletely processed precursor form of the lysosomal enzyme.

Secretion of a cysteine proteinase from a hormone-independent cell population of cultured explants of murine mammary gland.

Cultured explants of mouse mammary gland were investigated for their capacity to secrete a cathepsin B-like cysteine proteinase. This enzyme had been shown previously to be secreted excessively from cultured explants of human breast tumors and spontaneous mouse mammary carcinomas. We now show that secretion is also observed from cultured explants of mammary gland. Lactating tissue and tissues obtained from mid- and late-pregnant mice were found to secrete the cysteine proteinase at very high rates, but secretion was also detectable from explants of virgin mammary glands and from tissues obtained from retired breeders. However, in all cases, it was found that secretion was greatest from explants maintained in hormone-free medium and did not depend on the maintenance of normal mammary gland function. Secretion was greatly reduced in the presence of the lactogenic hormone combination of insulin, prolactin, and hydrocortisone, and this suppression was found to be due to hydrocortisone. Insulin and prolactin, while resulting in better tissue maintenance in culture, had no effect on the secretion. Enzyme release was reversibly inhibited by cycloheximide and required the presence of viable tissue, ruling out the possibility that the accumulation of enzyme activity in the culture medium is due to dying cells. The presence of metabolically active cells in explants cultured in the absence of hormones was also demonstrated by the incorporation of radiolabeled precursors into protein and DNA. DNA synthesis in cultured explants of lactating tissue was not stimulated by the addition of hormones. Histological studies revealed that, while large areas of the explants showed severe degeneration after culture without added hormones, clusters of cells persisted which displayed a relatively high mitotic activity and which showed a lack of normal epithelial organization. These observations suggest the presence of a hormone-independent cell population in the mammary gland, which secretes the stable cysteine proteinase. The secretion process itself, however, is inhibited by corticosteroids.

Characterization of a cysteine proteinase secreted by mouse mammary gland.

The cysteine proteinase activity secreted by cultured mammary gland was characterized to determine its relationship to a similar enzyme secreted by explants of mouse mammary tumors. Enzymic characterization showed that the secreted enzyme was similar to the lysosomal cysteine proteinase cathepsin B, and physical characterization showed properties identical to a stable cysteine proteinase secreted from mammary tumors reported previously. The secreted enzyme cross-reacted with mouse cathepsin B isolated from liver in a radioimmunoassay. The secreted enzymes are stable at alkaline pH, but irreversible conformational changes can be induced in vitro which render them unstable and thus similar to lysosomal cathepsin B, which is also unstable at alkaline pH. Tissue homogenates from fresh and cultured mammary gland contain mainly pH-unstable cathepsin B; however, the molecular size for the tissue cathepsin B, while smaller than that of the secreted enzymes, was found to be larger than that reported for mouse liver cathepsin B. Isoelectric focusing profiles were also slightly different as compared to those of mouse liver. These data suggest that there might be differences in the processing of cathepsin B between different tissues and organs, and the high degree of similarity between the forms of cathepsin B secreted from mouse mammary tumors, mouse mammary gland, and human malignant breast tumors suggests a similar mechanism for their extracellular release in these tissues.

Extracellular presence of the lysosomal proteinase cathepsin B in rheumatoid synovium and its activity at neutral pH.

The presence of the lysosomal proteinases cathepsin B and cathepsin D at extracellular sites in rheumatoid synovium was demonstrated using the antibody capture technique. Unlike cathepsin D, the cysteine proteinase cathepsin B was commonly detected only at the edges of the synovial explants. Radioimmunoassay and enzyme activity assay of these proteinases demonstrated that both were released from rheumatoid synovial cells in comparable amounts. Since lysosomal cathepsin B is unstable and denatured at physiologic pH and the antibody used only recognizes inactivated enzyme, we believe the selective detection of cathepsin B at the edge of the synovium may be due to the proteinase maintaining a native conformation within the explant, where the pH may be low enough to permit this. By use of a fluorescent substrate in a sensitive, continuous enzyme assay, cathepsin B was shown to express significant activity at neutral and alkaline pH before being inactivated. This and earlier work from this laboratory indicate that cathepsin B secreted by rheumatoid synovial cells may possess extracellular activity in vivo and be involved in the degradation of connective tissue macromolecules.

Characterization of a latent cysteine proteinase from ascitic fluid as a high molecular weight form of cathepsin B.

The latent cysteine proteinase present in ascitic fluid of patients with neoplasia and released from ascites cells in culture has been partially purified and the enzyme after pepsin activation was shown to be immunologically related to the lysosomal proteinase, cathepsin B. The latent form was characterized as a single chain of Mr 40 000 as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by Western blotting and immune staining with an antiserum to human cathepsin B. Using the same techniques the enzyme after pepsin activation gave a single band of Mr 33 000. Analysis by isoelectric focusing showed that the latent enzyme before and after pepsin treatment is composed of several acidic isoenzymes. These findings suggest that this latent proteinase represents a precursor form of cathepsin B which is released extracellularly rather than being processed and directed to the lysosome.